The physiological effects of cyclic AMP in mammalian tissues are thought to be mediated by the action of cyclic AMP-dependent protein kinases. Mechanistic studies are being carried out in our laboratory on bovine cardiac muscle protein kinase, a cyclic AMP-activated enzyme which catalyzes phosphate transfer from ATP to peptide and protein substrates. In most of our work we have used the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly and related peptides in which the residues on either side of the Ser residue have been replaced by other amino acids. Our current studies include an investigation of the identity of the inhibitory metal ion binding site on the catalytic subunit, the determination of the timing of the transfer of the metal ion-nucleotide complex from the gamma-phosphoryl group to the alpha-phosphoryl moiety, the characterization of important nucleophilic groups in the active site, and establishment of the stereochemistry of the binding of cyclic AMP to the regulatory subunit. Our research should provide much useful information about the interaction of cyclic AMP with isolated enzymes.